The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase Cα and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.
Bibliographical noteFunding Information:
This work was supported in part by the American Heart Association Scientist Development Grant 13SDG14270009 and National Institutes of Health Grants 1DP2 CA186752-01 and 1-R01-GM-105646-01-A1 (to S. S.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.